Topic A) Ca2+ influx channels (35% effort) Project 1: Translocation between PI(4,5)P2-poor and PI(4,5)P2-rich microdomains during store depletion determines STIM1 conformation and Orai1 gating. The Orai1 channel is the pore forming subunit that is activated by the ER Ca2+ sensor STIM1. Ca2+ influx by Orai1 mediates virtually all cellular functions from gene activation to exocytosis on time scales of msec to days. However, aberrant over activation of Orai1 and likely the TRPC channels occurs in many diseases and most often is the trigger of the disease as occurs in pancreatitis and Sjgren's syndrome. The activity of these channels is tightly regulated by the entering Ca2+ itself and is mediated by the STIM1 regulator SARAF. Preliminary data in the lab shows that it is the regulation by SARAF that becomes abnormal in disease states associated with excessive Ca2+ influx. We therefore study the molecular mechanism by which SARAF regulates Ca2+ influx and how it is altered in disease states. This led us to discovered that the regulation by SARAF takes place at the ER/PM junctions and thus we now study the formation of the junctions by tether proteins as it relates to the activity of the Ca2+ influx channels. We used of slow Ca2+-dependent inactivation (SCDI) of Orai1 by SARAF as a probe of the conformation and microdomain localization of the Orai1-STIM1 complex. We found that interaction of STIM1 with Orai1 C terminus and the STIM1 K-domain are required for interaction of SARAF with STIM1 and SCDI. STIM1-Orai1 must be in a PM/ER junctions tethered by E-Syt1, stabilized by Septin4 and enriched in PI(4,5)P2 for STIM1-SARAF interaction. Targeting STIM1 to PI(4,5)P2 rich and poor microdomains reveals that SARAF-dependent SCDI is observed only when STIM1-Orai1 are within the PI(4,5)P2-rich microdomain. Notably, store depletion results in transient localization of STIM1-Orai1 in the PI(4,5)P2-poor microdomain, which then translocate to the PI(4,5)P2-rich domain. These findings reveal the role of PM/ER tethers in the regulation of Orai1 function and a new mode of regulation by PI(4,5)P2 involving translocation between PI(4,5)P2 microdomains, rather than by synthesis and breakdown of PI(4,5)P2. These studies have been published in Nature Communication. Topic B) Intracellular Ca2+ channels (15% effort) Project 2: Regulation of the lysosomal two-pore channel-1 by Mg&#8314;, NAADP, PI(3,5)P&#8322;. Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. In a previous study, we provided conclusive evidence that TPC2 is NAADP-activated channel and further discovered that TPC2 is a Mg2+ and energy sensor. Since the endolysosomal system is the hub for energy sensing both through mTOC1 and, based on our work, Mg2+ sensing, and the endolysosomes also express TPC1, we are determining whether TPC1 is activated by NAADP and PI(3,5)P2, how the activation by NAADP and PI(3,5)P2 are related and whether and how TPC1 is regulated by Mg2+. Topic C) fluid and HCO3- secretion (35% effort) Project 3: Intracellular Cl- as a Signaling ion that Potently Regulates Na+/HCO3- Transporters. Introduction & Significance: A major topic in the lab is studying the transport function and role of members of the SLC26 transporters superfamily, CFTR and the Na+-HCO3- cotransporters NBCs. In addition to the transport of HCO3-, many of the transporters are transport Cl- or are exposed to large changes in Cl- concentration during fluid and electrolyte transport. An example, are the ducts of secretory glands like the salivary glands and the pancreas, in which Cl-in is reduced from 40-50 mM in the resting state to 5-10 mM in the stimulated secretory state. How cells sense and respond to changes in Cl-in is not known. We discovered that Cl-in functions as a signaling ion by regulation the activity of the basolateral 2Na+-1HCO3- cotransporter NBCe1-B and likely other Cl- and HCO3- transporters. Cl- is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl- (Cl-in) as a signaling ion has not been previously evaluated. We found that Cl-in functions as a regulator of cellular Na+ and HCO3- concentrations and transepithelial transport through modulating the activity of several electrogenic Na+-HCO3- transporters. We described the molecular mechanism of this regulation by physiological Cl-in concentrations, highlighting the role of GXXXP motifs in Cl- sensing. Regulation of the ubiquitous NBCe1-B is mediated by two GXXXP containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl-in interacting at a low affinity GXXXP-containing site. IRBIT activation of NBCe1-B unmasks a second high affinity Cl-in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl-in interacting site. NBCe1-A is unaffected by Cl-in between 5-140 mM. However, deletion of NBCe1-A residues 29-41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl-in. These findings reveal a novel cellular Cl-in sensing mechanism that plays an important role in the regulation of Na+ and HCO3- transport with critical implications for the role of Cl- in cellular ion homeostasis and epithelial fluid and electrolyte secretion. These studies were published in The PNAS.